1. Field of the Invention
The present invention relates to an apparatus for performing biological reactions on a substrate surface and a method for removing gas bubbles from the apparatus. Specifically, the invention relates to an apparatus having a flexible, gas permeable layer affixed to a substrate layer with an adhesive, wherein the flexible, gas permeable layer, the adhesive and the substrate layer enclose a reaction chamber, and a means for facilitating diffusion across the flexible, gas permeable layer. The diffusion-facilitating means creates a pressure gradient or concentration gradient across the flexible, gas permeable layer, thereby increasing the rate of diffusion of gas molecules from the reaction chamber across the flexible, gas permeable layer.
2. Description of the Prior Art
Recent advances in molecular biology have provided the opportunity to identify pathogens, diagnose disease states, and perform forensic determinations using gene sequences specific for the desired purpose. This explosion of genetic information has created a need for high-capacity assays and equipment for performing molecular biological assays. Most urgently, there is a need to miniaturize, automate, standardize and simplify such assays. While these assays were originally developed in research laboratories working with purified products and performed by highly skilled individuals, adapting these procedures to clinical uses, such as diagnostics, forensics and other applications, has produced the need for equipment and methods that allow less-skilled operators to effectively perform the assays under higher capacity, less stringent assay conditions.
Existing technology utilizes the binding of molecules contained within a biologically reactive sample fluid, hereinafter referred to as target molecules, onto molecules contained within biologically reactive sites, hereinafter referred to as probe molecules. The primary enabler of this technology is an apparatus commonly referred to as a biochip, which comprises one or more ordered microscopic arrays (xe2x80x9cmicroarraysxe2x80x9d) of biologically reactive sites immobilized on the surface of a substrate. A biologically reactive site can be created by dispensing a small volume of a fluid containing a biological reagent onto a discrete location on the surface of a substrate, also commonly referred to as spotting. To enhance immobilization of probe molecules, biochips can include a 2-dimensional array of 3-dimensional polymeric anchoring structures (for example, polyacrylamide gel pads) attached to the surface of the substrate. Probe molecules such as oligonucleotides are covalently attached to polyacrylamide-anchoring structures by forming amide, ester or disulfide bonds between the biomolecule and a derivatized polymer comprising the cognate chemical group. Covalent attachment of probe molecules to such polymeric anchoring structures is usually performed after polymerization and chemical cross-linking of the polymer to the substrate is completed.
Biochips are advantageously used to perform biological reactions on the surface thereof. Existing apparatus for performing biological reactions on a substrate surface, however, are deficient in that they either require unacceptably large volumes of sample fluid to operate properly, cannot accommodate substrates as large as or larger than a conventional microscope slide, cannot independently accommodate a plurality of independent reactions, or cannot accommodate a substrate containing hydrogel-based microarrays. Most existing apparatus also do not allow introduction of fluids in addition to the sample fluid (such as wash buffers, fluorescent dyes, etc.) into the reaction chamber. Disposable apparatus must be disassembled and reassembled around the biochip every time a new fluid must be introduced. Other existing apparatus are difficult to use in a laboratory environment because they cannot be loaded with standard pipet tips and associated pipettor apparatus.
Many existing apparatus also exhibit unacceptable reaction reproducibility, efficiency, and duration. Reaction reproducibility may be adversely affected by bubble formation in the reaction chamber or by the use of biologically incompatible materials for the reaction chamber. Reaction duration and efficiency may be adversely affected by the presence of concentration gradients in the reaction chamber.
Bubbles can form upon introduction of sample fluid to the reaction chamber or by outgassing of the reaction chamber materials. When gas bubbles extend over the substrate surface in an area containing biologically reactive sites, the intended reaction may intermittently fail or yield erroneous results because the intended concentration of the sample fluid mixture has been compromised by the presence of gas bubbles.
Biologically incompatible reaction chamber materials may cause unacceptable reaction reproducibility, by interacting with the sample fluid, thus causing the intended reaction to intermittently fail or yield erroneous results.
Incomplete mixing of the sample fluid can introduce concentration gradients within the sample fluid that adversely impact reaction efficiency and duration. This effect is most pronounced when there is a depletion of target molecules in the local volume surrounding a biologically reactive site. During a biological reaction, the probability that a particular target molecule will bind to a complementary (immobilized) probe molecule is determined by the given concentration of target molecules present within the sample fluid volume, the diffusion rate of the target molecule through the reaction chamber, and the statistics of interaction between the target molecule and the complementary probe molecule. For diagnostic assays, target DNA molecules are often obtained in minute ( less than picomol) quantities. In practice, it can take tens of hours for a hybridization reaction to be substantially complete at the low target nucleic acid molecule levels available for biological samples. Concentration gradients in the hybridization chamber can further exacerbate this problem.
U.S. Pat. No. 5,948,673 to Cottingham discloses a self-contained multi-chamber reactor for performing both DNA amplification and DNA probe assays in a sealed unit wherein some reactants are provided by coating the walls of the chambers and other reactants are introduced into the chambers prior to starting the reaction in order to eliminate flow into and out of the chamber. No provisions are made for eliminating gas bubbles from the chambers.
There remains a need in the art for methods and apparatus for performing biological reactions on a substrate surface that use a low volume of sample fluid, that accommodate substrates as large as or larger than a conventional microscope slide, that accommodate a plurality of independent reactions, and that accommodate a substrate surface having one or more hydrogel-based microarrays attached thereto. There also remains a need in the art for an apparatus that allows introduction of fluids in addition to sample fluid into each reaction chamber via standard pipet tips and associated pipettor apparatus. There also remains a need in the art for such an apparatus that increases reaction reproducibility, increases reaction efficiency, and reduces reaction duration. There also remains a need in this art for a simple method for removing gas bubbles from such an apparatus. These needs are particularly striking in view of the tremendous interest in biochip technology, the investment and substantial financial rewards generated by research into biochip technology, and the variety of products generated by such research.
The invention provides an apparatus for performing biological reactions on a substrate surface and a method for removing gas bubbles from the apparatus to prevent interference with biological reactions such as hybridization at reaction sites on-the substrate surface. Specifically, the method of the invention is directed to an apparatus comprising a flexible, gas permeable layer affixed to a biochip with an adhesive, wherein the flexible, gas permeable layer, the adhesive, and the biochip enclose a reaction chamber, and a means for facilitating diffusion of gas molecules out of the reaction chamber across the flexible, gas permeable layer. The diffusion-facilitating means creates a pressure gradient or concentration gradient across the flexible, gas permeable layer, thereby increasing the rate of diffusion of gas molecules from the reaction chamber through the flexible, gas permeable layer.
The biochip comprises a substrate having a first surface and a second surface, wherein the first surface contains an array of biologically reactive sites, and is preferably an oligonucleotide array. The array is provided in an area bounded by an adhesive set down on the first substrate surface. The flexible, gas permeable layer, the adhesive and the first substrate surface further define a volume comprising a reaction chamber.
The flexible, gas permeable layer preferably is deformable, translucent, and porous. More preferably, the flexible, gas permeable layer is selectively permeable to gas but impermeable to liquid. Most preferably, the flexible, gas permeable layer is selectively permeable to gases and impermeable to liquids because the surface tension of the sample fluid prevents escape of the liquid through the pores of the flexible membrane.
In certain embodiments of the invention, the substrate comprises a multiplicity of oligonucleotide arrays, which are contained in one or a plurality of areas bounded by the adhesive and covered by the flexible, gas permeable layer.
Each of the reaction chambers also preferably include a first port, and certain embodiments further include a second port, that transverses the substrate and comprises a first opening on the first substrate surface and a second opening on the second substrate surface. The openings of these ports on the second substrate surface are covered by a removable cover, most preferably a foil tape. The openings of these ports on the first substrate surface are provided within the area bounded by the adhesive.
The adhesive, the flexible, gas permeable layer and the substrate also enclose a reaction chamber that is filled prior to use with a water-soluble compound. The water-soluble compound is preferably a solid at a temperature most preferably at or below room temperature, and a liquid at higher temperatures, most preferably below about 100xc2x0 C.
In preferred embodiments, the diffusion-facilitating means creates a pressure differential across the flexible, gas permeable layer. In more preferred embodiments, the diffusion-facilitating means comprises a vacuum source removably affixed to the flexible, gas permeable layer, wherein the vacuum source is used to apply a vacuum to the flexible, gas permeable layer. Most preferably, the vacuum source comprises a vacuum pump connected by a length of plastic tubing to a reducer that completely encloses the area defined by the adhesive and is removably sealed to the flexible, gas permeable layer.
The chamber is also optionally supplied with a roller, most preferably a patterned roller, positioned in contact with the flexible, gas permeable layer and movable longitudinally across the surface of the chamber for mixing sample fluid and wash solutions as required.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.